CN111172008B

CN111172008B - Method and device for automatic nucleic acid extraction

Info

Publication number: CN111172008B
Application number: CN201811330464.3A
Authority: CN
Inventors: 李德政
Original assignee: Catchgene Co ltd
Current assignee: Catchgene Co ltd
Priority date: 2018-11-09
Filing date: 2018-11-09
Publication date: 2021-09-28
Anticipated expiration: 2038-11-09
Also published as: WO2020093824A1; EP3878939A1; CN111172008A; US20220017890A1; JP7141530B2; JP2022506567A; EP3878939A4

Abstract

The invention provides a method and a device for automatically extracting nucleic acid. The automatic nucleic acid extracting device comprises a seat body, a cassette, a driving unit, a moving frame and a needle cylinder. The base body is provided with a sample accommodating area, a pipe column accommodating area, a cassette accommodating area and a collecting pipe, and the sample accommodating area, the pipe column accommodating area, the cassette accommodating area and the collecting pipe are arranged along a linear direction. The cartridge is arranged in the cartridge accommodating area and comprises two parallel walls and at least two vertical walls, the parallel walls and the vertical walls jointly form a dissolving liquid tank, a washing liquid tank and an eluent liquid tank, wherein the vertical walls forming the dissolving liquid tank, the washing liquid tank and the eluent liquid tank are respectively provided with an abutting part, and the dissolving liquid tank, the washing liquid tank and the eluent liquid tank are arranged along a linear direction. The abutting portion has a circular arc wall or a polygonal wall. The driving unit and the movable frame are arranged on the base body. The needle cylinder is arranged on the moving frame and driven by the driving unit to reciprocate along the linear direction along with the moving frame. The automatic nucleic acid extraction device can quickly and conveniently extract nucleic acid with higher yield and higher concentration from a sample.

Description

Method and device for automatic nucleic acid extraction

Technical Field

The present invention relates to an apparatus and a method, and more particularly, to an automated nucleic acid extraction apparatus and an automated nucleic acid extraction method.

Background

With the innovation of human genome sequencing technology, the advancement of biomedical analysis technology, and the emergence of big data analysis tools, the era of Precision Medicine (Precision Medicine) has come. Accurate medical treatment is a customized medical treatment mode, which is based on human genome information and combined with related internal environment information such as proteome, metabolome and the like to formulate an optimal treatment scheme for patients in order to maximize the treatment effect and minimize side effects.

Blood is a red opaque viscous liquid flowing in blood vessels and heart of human body, and its main components are blood plasma, blood cells, and genetic materials (chromosomes and genes). Among them, cell free DNA (cfDNA) is an isolated DNA fragment found in plasma, and circulating tumor DNA (ctDNA) fragment refers to a DNA fragment released from a tumor in cfDNA.

Almost everyone has free DNA fragments (i.e. cfDNA) in the blood, which may originate from apoptosis, necrosis of cells, or enter the blood by means of active release (vigorous movement). However, the cfDNA content in blood is very low, between about 1ng to 100ng cfDNA in 1mL plasma, and even lower, only between 0.1% to 5% of cfDNA.

Researches have proved that the total amount of cfDNA in peripheral blood of a tumor patient is higher than that of a healthy person, and by the fact that the cfDNA content is increased, a good prompting effect can be achieved, and therefore the cfDNA content can be used as a means for primary screening of tumors. Therefore, the purification of a trace amount of cfDNA (a kind of nucleic acid) from a Liquid biological sample (Liquid Biopsy) is the first step of precision medicine.

Researchers in the field have been working on developing devices and methods for extracting nucleic acids such as cfDNA or ctDNA. For example, CN101684463A provides a method for rapidly extracting nucleic acids from various trace clinical samples, which comprises the following steps: (a) adding the clinical sample into the lysis solution, and uniformly mixing; (b) sucking a clinical sample, allowing the sample to flow through a filter membrane, adsorbing nucleic acid components in the sample on the filter membrane due to specific adsorption of the membrane, and allowing filtrate to be waste liquid containing cell debris and protein; (c) pumping the cleaning solution, allowing the cleaning solution to flow through the filtering membrane, cleaning residual protein or other components on the filtering membrane, and discarding the filtrate; (d) the eluate is aspirated and passed through a filtration membrane to elute the nucleic acid component adsorbed on the membrane, thereby obtaining an aqueous nucleic acid solution containing no other impurities for nucleic acid amplification.

TWM477925U provides a sample extraction device comprising a take-up member comprising a first upper portion, an intermediate portion communicating below the first upper portion and having an inner diameter smaller than the inner diameter of the first upper portion, and a take-up portion communicating below the intermediate portion and having an inner diameter smaller than the inner diameter of the intermediate portion, whereby the first upper portion or the intermediate portion fits against the outer periphery of the column member and takes up a specific liquid to the column member via the take-up portion; the sample extraction device further comprises: a tubular string component; and a means for receiving the sample, thereby extracting the desired material from the sample.

TWM536238U provides an automated nucleic acid extraction apparatus, comprising: the tray fixing frame can move along a horizontal rail; the bracket is vertically arranged above the machine table bottom plate and is provided with a vertical track; the vertical motion unit comprises a substrate, a substrate rail is arranged above the substrate, a motion block is arranged along the substrate rail, and a syringe fixing unit which is positioned below the motion block and is fixedly locked on the substrate is used for a syringe to be erected on; and the base plate is arranged along the vertical track and can drive the moving block and the needle cylinder fixing unit to move up and down corresponding to the bracket.

TW201412981A provides a method and apparatus for extracting nucleic acid using a pneumatic method, in which a positive or negative gas pressure device is connected to the upper end of a purification tube, so that the sample, the washing solution and the washing solution can be sucked/discharged from the lower tip of the purification tube, thereby achieving the effect of extracting nucleic acid easily without using a centrifuge.

Although the above prior arts have been described, these prior arts still have a disadvantage that cfDNA or ctDNA cannot be extracted in large quantities. Therefore, if an automated nucleic acid extraction apparatus capable of extracting cfDNA or ctDNA in large quantities to improve the yield could be developed, it would bring a breakthrough to precision medicine.

Disclosure of Invention

The invention aims to provide an automatic nucleic acid extraction device and an automatic nucleic acid extraction method. Compared with the prior art, the automatic nucleic acid extraction device and method of the invention can rapidly and conveniently extract nucleic acid with higher yield and higher concentration, such as cfDNA and ctDNA, from a sample.

The present invention provides an automatic nucleic acid extracting apparatus, which comprises a base, a cassette, a driving unit, a moving frame and a syringe. The base body is provided with a sample accommodating area, a pipe column accommodating area, a cassette accommodating area and a collecting pipe, and the sample accommodating area, the pipe column accommodating area, the cassette accommodating area and the collecting pipe are arranged along a linear direction. The cartridge is arranged in the cartridge accommodating area and comprises two parallel walls and at least two vertical walls, the parallel walls and the vertical walls jointly form a dissolving liquid tank, at least one washing liquid tank and an eluent liquid tank, wherein the vertical walls forming the dissolving liquid tank, the washing liquid tank and the eluent liquid tank are respectively provided with an abutting part, and the dissolving liquid tank, the washing liquid tank and the eluent liquid tank are also arranged along the linear direction, wherein the abutting part is provided with an arc-shaped wall or a polygonal wall. The driving unit is arranged on the seat body. The moving frame is vertically arranged on the seat body and driven by the driving unit to reciprocate along the linear direction. The needle cylinder is arranged on the movable frame and moves along with the movable frame.

In one embodiment, the sample holding area has a sample holding space and a binding buffer holding space.

In one embodiment, the column receiving area further includes a sample row straw receiving space and a column row straw receiving space.

In one embodiment, the automated nucleic acid extracting apparatus further comprises a sample row pipette movably disposed in the sample row pipette accommodating space.

In one embodiment, the automated nucleic acid extraction apparatus further comprises a column row pipette movably disposed in the column row pipette receiving space.

In one embodiment, the syringe is removably attached to the sample or column discharge pipette.

In an embodiment, the sample accommodating space is used for accommodating a biological sample and a dissolution buffer solution for performing a dissolution reaction, the combination buffer solution accommodating space is used for accommodating a combination buffer solution and a dissolution product for performing a combination reaction, the dissolution liquid tank is used for accommodating a dissolution buffer solution, an antifoaming agent and a reaction residue, the washing liquid tanks are used for accommodating a washing buffer solution, the elution liquid tanks are used for accommodating an elution buffer solution, the sample exhaust and suction pipe accommodating space is used for accommodating a sample exhaust and suction pipe, and the column exhaust and suction pipe accommodating space is used for accommodating a column exhaust and suction pipe.

In one embodiment, the arc angle of the radiused wall is greater than or equal to 90 degrees.

In one embodiment, the vertical wall between the wash solution tank and the eluent solution tank has a complete hollow cylindrical structure, forming a filter accommodating space for accommodating a filter.

In one embodiment, the polygonal wall comprises at least two supporting walls, and the supporting walls mutually form an included angle of less than 180 degrees.

In one embodiment, the bottom of the eluate tank forms a recess.

In one embodiment, the cartridge further comprises an elastic fastener for detachably fastening the cartridge in the cartridge accommodating area.

The present invention also provides an automated nucleic acid extraction method using the automated nucleic acid extraction apparatus as described above, comprising the steps of: sucking the mixed reactant in the buffer solution containing space by matching the needle cylinder with a column suction pipe, so that the nucleic acid in the reactant is combined to a diaphragm in the column suction pipe, and discharging the reaction residue to a cassette so that the nucleic acid in the reaction residue is combined to the diaphragm; absorbing the washing buffer solution in at least one washing liquid tank through a diaphragm by matching the needle cylinder with the column suction pipe, and then discharging the washing buffer solution through the diaphragm by matching the needle cylinder with the column suction pipe; and sucking the elution buffer solution in the elution liquid groove by using the needle cylinder and the column suction tube through the membrane, and then discharging the elution buffer solution with nucleic acid to the collection tube by using the needle cylinder and the column suction tube through the membrane.

In one embodiment, in the step of discharging the reaction residue into the cassette and the step of discharging the washing buffer from the syringe together with the column pipette through the membrane, the abutting portion of the column pipette is abutted against the abutting portion of the cassette, and then the reaction residue or the washing buffer is discharged.

In one embodiment, the method for automated nucleic acid extraction further comprises the following steps after the steps of abutting the abutting portion of the pipette against the abutting portion of the cassette and then discharging the reaction residue or the washing buffer: the abutting part of the sucker in the column row abuts against the abutting part of the cassette, and the needle cylinder moves up and down along the direction vertical to the linear direction by less than or equal to 5 mm.

In summary, the invention has the following effects: the parallel walls and the vertical walls of the cartridge are arranged to form a solution tank, a washing solution tank and an eluent tank which can hold large volumes of solution; and through the design that the washing liquid groove and the eluent groove are respectively provided with the abutting parts, after the needle cylinder discharges large-volume solution, the sample discharging suction pipe or the column discharging suction pipe can be prevented from falling off from the needle cylinder and separating from the needle cylinder. In addition, through the design that the cassette and the accommodating space are arranged along the linear direction and the moving frame and the needle cylinder reciprocate along the linear direction, the automatic nucleic acid extraction device can achieve the effect of automatically extracting nucleic acid in the linear direction, avoid the pollution of a sample and improve the extraction efficiency, so the automatic nucleic acid extraction device can really extract nucleic acid with higher yield and higher concentration, such as cfDNA and ctDNA, from the sample quickly and conveniently. Furthermore, compared with the prior art, the cassette and the accommodating space move linearly (i.e. the cassette moves and the needle cylinder does not move relative to the table), the present invention moves linearly (i.e. the needle cylinder moves and the cassette does not move relative to the table), so the automatic nucleic acid extracting apparatus of the present invention has a smaller operation space, which is about twice as long as the cassette and the accommodating space, and the prior art moves linearly from the cassette and the accommodating space, which is about 2 to 3 times as long as the cassette and the accommodating space. Therefore, the automatic nucleic acid extraction device of the invention has smaller volume and can save the use space of users.

Drawings

FIG. 1 is a schematic diagram of an automated nucleic acid extraction apparatus according to a preferred embodiment of the present invention.

FIG. 2 is a side view of an automated nucleic acid extraction apparatus according to a preferred embodiment of the present invention.

FIG. 3A is a partial schematic view of an automated nucleic acid extraction apparatus according to a preferred embodiment of the present invention.

FIG. 3B is a partial schematic view of an automated nucleic acid extraction apparatus according to a preferred embodiment of the present invention.

FIG. 3C is a partial schematic view of an automated nucleic acid extraction apparatus according to a preferred embodiment of the present invention.

FIG. 4 is a partial schematic view of an automated nucleic acid extraction apparatus according to a preferred embodiment of the present invention.

FIG. 5A is a partial schematic view of an automated nucleic acid extraction apparatus according to a preferred embodiment of the present invention.

FIG. 5B is a partial schematic view of an automated nucleic acid extraction apparatus according to a preferred embodiment of the present invention.

FIG. 5C is a partial schematic view of an automated nucleic acid extraction apparatus according to a preferred embodiment of the present invention.

FIG. 6A is a top view of a cassette of an automated nucleic acid extraction apparatus according to a preferred embodiment of the present invention.

FIG. 6B is a top view of another preferred embodiment of the cassette of the automated nucleic acid extracting apparatus according to the present invention.

FIG. 6C is a top view of another preferred embodiment of the cassette of the automated nucleic acid extracting apparatus according to the present invention.

FIGS. 6D to 6H are schematic views of different embodiments of the abutting portion of the cassette of the automated nucleic acid extracting apparatus according to the present invention.

FIG. 7A is a partial enlarged view of the automated nucleic acid extraction apparatus of FIG. 1.

FIG. 7B is a schematic diagram of the automated nucleic acid extraction apparatus of FIG. 7A.

FIG. 7C is another schematic diagram of the automated nucleic acid extraction apparatus of FIG. 7A.

FIG. 8A is a schematic diagram of a portion of the automated nucleic acid extraction apparatus of FIG. 1A.

FIG. 8B is an exploded view of the automated nucleic acid extraction apparatus of FIG. 8A.

FIG. 9 is a flow chart of an automated nucleic acid extraction method according to a preferred embodiment of the present invention.

Detailed Description

The following description will be made with reference to the accompanying drawings, in which like elements are designated by like reference numerals, illustrating preferred embodiments of an automated nucleic acid extracting apparatus according to the present invention.

The automatic nucleic acid extraction device can quickly and conveniently extract nucleic acid with higher yield and higher concentration, such as cfDNA and ctDNA, from a sample. In particular, samples include, but are not limited to: blood, plasma, urine, saliva, interstitial fluid and tissue. The following will illustrate the structure and features of an automated nucleic acid extraction apparatus by way of example.

Please refer to fig. 1 and 2, wherein fig. 1 is a schematic combination diagram of an automated nucleic acid extraction apparatus according to a preferred embodiment of the present invention, and fig. 2 is a side view of an automated nucleic acid extraction apparatus according to a preferred embodiment of the present invention.

As shown in FIGS. 1 and 2, the automated nucleic acid extracting apparatus includes a housing B, a cassette 31, a driving unit M, a moving frame 4 and a syringe 5.

The base body B has a sample accommodating area 1, a column accommodating area 2, and a cassette accommodating area 3, the sample accommodating area 1 has a sample accommodating space 11 and a combined buffer accommodating space 12, and the sample accommodating area 1, the column accommodating area 2, and the cassette accommodating area 3 are arranged along a linear direction L1. Specifically, the sample holding region 1, the column holding region 2 and the cassette holding region 3 are arranged along a linear direction L1 (from right to left in the direction of the drawing, the same below) in the sequence such as, but not limited to, the sample holding region 1, the column holding region 2 and the cassette holding region 3; firstly, a tubular column accommodating area 2, a cassette accommodating area 3 and a sample accommodating area 1; firstly, a cassette accommodating area 3, a sample accommodating area 1 and a column accommodating area 2; or the cassette accommodating area 3, the column accommodating area 2 and the sample accommodating area 1.

Referring to fig. 2 and fig. 6A, the cassette 31 is disposed in the cassette receiving area 3, the cassette 31 includes two parallel walls 31a and at least two vertical walls 31b, the parallel walls 31a and the vertical walls 31b form a solution tank 311, at least one washing solution tank 312 and an eluent tank 313, wherein the vertical walls 31b forming the solution tank 311, the washing solution tank 312 and the eluent tank 313 are respectively provided with an abutting portion 314, and the solution tank 311, the washing solution tank 312 and the eluent tank 313 are also arranged along a linear direction L1. In the present embodiment, the number of the vertical walls 31b is 9, and the vertical walls 31b and the parallel walls 31a together form 2 dissolution

liquid tanks

311, 4 washing liquid tanks 312 and 2 eluent liquid tanks 313, however, the number of the vertical walls 31b, the dissolution liquid tanks 311, the washing liquid tanks 312 and the eluent liquid tanks 313 can be adjusted by the user according to the actual needs (for example, if the sample is a tissue that is difficult to extract, the number of the dissolution liquid tanks 311 and the eluent liquid tanks 313 can be increased), and the invention is not limited thereto. In addition, the solution tank 311, the washing tank 312, and the eluent tank 313 formed by the arrangement of the parallel walls 31a and the vertical walls 31b of the cassette 31 can accommodate a large volume of buffer (dissolution buffer, washing buffer, and/or elution buffer), such as, but not limited to, 2ml or more, 5 ml or more, or 10 ml or more.

Referring to fig. 1 and fig. 2 again, in the present embodiment, the driving unit M is disposed on the seat B, and the movable frame 4 is vertically disposed on the seat B and driven by the driving unit M to reciprocate along the linear direction L1. The needle cylinder 5 is provided to the movable frame 4 and moves with the movable frame 4. In particular, although the driving unit M is shown as a transmission roller belt, the driving unit M may be other driving devices, such as a linear module or other devices capable of driving the moving frame 4 to move linearly, and the invention is not limited thereto.

Referring to fig. 1, 2 and 4, in the present embodiment, the column accommodating section 2 further includes a sample exhaust pipe accommodating space 21 and a column exhaust pipe accommodating space 22.

Referring to FIG. 4, in the present embodiment, the automated nucleic acid extracting apparatus further includes a sample row pipette 6 movably disposed in the sample row pipette accommodating space 21. In this embodiment, the automated nucleic acid extracting apparatus further comprises a column pipette 7 movably disposed in the column pipette receiving space 22. In particular, the syringe 5 is detachably connected to the sample discharge pipette 6 or the column discharge pipette 7.

Referring to FIGS. 1 to 3A, the automated nucleic acid extracting apparatus further includes a collection tube 8, and the cassette 31 and the receiving areas are arranged along a linear direction L1. In a preferred embodiment, the collection tube 8 is disposed adjacent to the cassette 31 (but the invention is not limited thereto), since the automated nucleic acid extraction method is to extract along the linear direction L1 and then discharge the eluate into the collection tube 8 in the final step, the collection tube 8 is disposed adjacent to the cassette 31 and arranged along the linear direction L1, which is the same as the moving direction of the movable rack 4, so as to prevent the movable rack 4 from passing over the collection tube 8 during the automated nucleic acid extraction process, thereby causing cross contamination.

In the present embodiment, the sample accommodating space 11 is used for accommodating a biological sample (biological sample) and a lysis buffer (lysis buffer) for performing a lysis reaction (lysis reaction). In this embodiment, the biological sample includes, but is not limited to: blood, plasma, urine, saliva, interstitial fluid and tissue. The binding buffer accommodating space 12 is used for accommodating a binding buffer and a lysate (lysate) to perform a binding reaction. The lysis solution tank 311 is used for accommodating lysis buffer (lysis buffer), defoaming agent (defoaming agent), and reaction residue. These washing liquid tanks 312 are used for holding washing buffer (wash buffer). These eluent liquid tanks 313 are for containing an elution buffer (elution buffer). The sample row pipette receiving space 21 is for receiving the sample row pipette 6. The column row suction pipe accommodating space 22 is for accommodating the column row suction pipes 7. In the present embodiment, the sample accommodating space 11, the binding buffer accommodating space 12, the lysis buffer tank 311 and the washing solution tank 312 can accommodate, for example, but not limited to, 30mL of sample or buffer solution, so that compared with the prior art (usually 2mL) having a higher sample capacity, the volume increase of the lysis buffer can improve the lysis reaction of the sample to increase the concentration of the nucleic acid; the volume of the washing buffer solution is increased to wash the solution remained in the column pipette 7, so as to improve the concentration and purity of the nucleic acid extracted in the subsequent steps. In the present embodiment, the number of the solution tank, the washing tank and the eluent tank can be adjusted according to the actual requirement of the user, and the present invention is not limited thereto.

Referring to fig. 3C, a diaphragm 74 is disposed inside the column suction pipe 7. The membrane 74 is made of, for example, but not limited to, a silica membrane (silica membrane), and has a positive charge, and during the nucleic acid extraction process of the sample, the nucleic acid contained therein can have a negative charge, and the nucleic acid can be attached to the membrane 74 by the property of attraction between the positive and negative charges, and then the nucleic acid attached to the membrane 74 can be extracted by the subsequent process.

In this embodiment, the seat body B further comprises at least one heating element H disposed below the sample accommodating space 11 and/or the pipestring accommodating space 22, so that when the heating element H is disposed below the sample accommodating space 11, the dissolution reaction between the biological sample and the dissolution buffer can be promoted, the biological sample can be dissolved more completely, and the concentration of the nucleic acid extracted by the subsequent process can be increased; when the heating member H is disposed below the tube row holding space 22, it can promote the volatilization of the residual solvent on the membrane 74 of the tube row 7, thereby increasing the concentration and purity of the nucleic acid extracted by the following procedure. In particular, when the nucleic acid to be extracted is ribonucleic acid (RNA), the heating element H under the sample holding space 11 may not be turned on (i.e., not heated during the dissolution reaction) to prevent the RNA from being decomposed and affecting the concentration of the extracted RNA.

Referring to fig. 3A and 3B, a recess f is formed at the bottom of the eluent tank 313. With this arrangement, a smaller amount of elution buffer can be used for elution of the sample, and the concentration of nucleic acid extraction can be increased. In particular, although only the eluate tank 313 adjacent to the collection tube 8 is shown to have the recess f, other eluate tanks 313 may have the recess f, and the present invention is not limited thereto. In addition, the volume of the elution buffer is not limited to 1ml, 500. mu.l, 200. mu.l, 100. mu.l, 50. mu.l, 30. mu.l, etc., and can be adjusted according to the user's needs, and the present invention is not limited thereto.

Referring to fig. 6A to 6C, the cassette 31 further includes an elastic fastener 316 for detachably fastening the cassette 31 to the cassette receiving area 3. Specifically, please refer to fig. 8A and 8B, wherein fig. 8A only shows a portion of the structure of the automated nucleic acid extracting apparatus of fig. 1, and fig. 8B is an exploded view of fig. 8A for easy understanding. The automated nucleic acid extraction apparatus of the present embodiment may further include an iron stand I detachably disposed on the base B. The cassettes 31 and the collecting pipes 8 can be set on the hob I by a user, and then set on the seat body B together with the hob I, so that the user can conveniently set the cassettes 31 and the collecting pipes 8. In addition, the user can also set the iron stand I on the seat B first, and then set the cassette 31 and the collecting pipe 8, which is not limited in the present invention. Furthermore, after extracting nucleic acid using the automated nucleic acid extracting apparatus, the rack I can be taken out from the seat B, so that a user can take out a plurality of cassettes 31 and collecting tubes 8 at a time. In particular, the iron stand I may include two handles I1 for facilitating the user to take the iron stand I out of the seat B or put the iron stand I into the seat B.

Referring to fig. 4, the sample pipette 6 includes a fitting portion 61 and a tip portion 62 connected to the fitting portion 61. The column pipette 7 comprises a fitting portion 71, an abutting portion 72, and a tip portion 73 abutting against the abutting portion 72, wherein the length D2 of the tip portion 73 is smaller than the depth D1 of the collection tube 8 (as shown in FIG. 3C). With this arrangement, when the tip portion 73 with nucleic acids attached thereto enters the collection tube 8, the sample with nucleic acids is prevented from flying out of the collection tube 8 due to the large force generated after the sample is discharged from the tip portion 73, thereby improving the yield of nucleic acid extraction. In the preferred embodiment, the fitting portion 61 and the tip portion 62 are integrally formed; the fitting portion 71, the abutting portion 72, and the tip portion 73 are integrally molded. Alternatively, the fitting portion 61 and the tip portion 62 may be detachable from each other, and the fitting portion 71, the abutting portion 72, and the tip portion 73 may be detachable from each other, but the present invention is not limited thereto. In particular, the sample exhaust pipette 6 may have the same configuration as the column exhaust pipette 7, and the difference between the two is that the column exhaust pipette 7 has the membrane 74, and the sample exhaust pipette 6 does not have the membrane 74 (that is, the sample exhaust pipette 6 may also have a fitting portion, an abutting portion, and a tip portion, and the sample exhaust pipette 6 and the column exhaust pipette 7 can be manufactured by using only one kind of mold, and the sample exhaust pipette 6 and the column exhaust pipette 7 are manufactured by taking out or putting in the membrane 74, respectively).

Referring to fig. 3B to 5B, the bottom of the syringe 5 further includes a joint 51, wherein the syringe 5 is connected to the mounting

portions

61 and 71 through the joint 51. In detail, the joint 51 is coupled with the structure of the fitting portion 61 of the sample discharge straw 6 or the fitting portion 71 of the column discharge straw 7 to tightly connect the syringe 5 with the

fitting portions

61, 71. In particular, the needle cylinder 5 and the adapter 51 may be integrally formed.

Referring to fig. 1 and fig. 7A to 7B, fig. 7A is an enlarged view of a region a in fig. 1. The movable frame 4 includes a material ejecting plate 41, and two sides of the movable frame 4 are respectively provided with a spring mechanism 42, the spring mechanism 42 drives the material ejecting plate 41 to operate to control the assembling

portions

61, 71 to be detached from the bottom of the syringe 5. Specifically, as shown in fig. 7B, the spring mechanism 42 moves in the direction L3, and drives the ejector plate 41 to move in the direction L4, so as to separate the sample pipette 6 or the column pipette 7 from the syringe 5.

Referring to fig. 7C in conjunction with fig. 7A, the movable frame 4 further includes at least one syringe fixing member 43 for fixing the syringe 5 on the movable frame 4. The syringe mount 43 may further include a syringe retaining groove 431 and at least one recess 432, and the recess 432 may be provided with an elastic member R. As shown in fig. 7A, the syringe 5 is fixed in the syringe fixing grooves 431 of the two syringe fixing members 43, and the syringe 5 is preferably fixed in the syringe fixing grooves 431 by the elastic members R provided in the concave holes 432. In particular, the number of the syringe fixing member 43, the concave hole 432 and the elastic member R may be adjusted according to the user's requirement, as long as the syringe 5 can be stably fixed in the syringe fastening groove 431, and the present invention is not limited thereto.

Referring to FIG. 1, FIG. 5A, FIG. 5B and FIG. 6A, wherein FIG. 5A and FIG. 5B are partial schematic views of an automated nucleic acid extraction apparatus according to a preferred embodiment of the present invention, and FIG. 6A is a top view of a cassette of the automated nucleic acid extraction apparatus according to a preferred embodiment of the present invention. As shown in fig. 5A, 5B and 6A, the abutting portion 314 has an arc-shaped wall 3141, and the arc angle θ of the arc-shaped wall 3141 is greater than or equal to 90 degrees. Preferably, the arc angle θ may be 90 degrees; preferably, the arc angle θ may be 120 degrees; preferably, the arc angle θ may be 180 degrees; preferably, the arc angle θ may be 270 degrees as long as the tubing string suction pipe 7 can abut against the arc angle. Specifically, as shown in fig. 6A, the vertical wall 31b has a circular arc-shaped wall 3141, and is connected to the parallel wall 31a by the vertical wall 31b, that is, both ends of the circular arc-shaped wall 3141 are not directly connected to the parallel wall 31 a.

Referring to FIG. 1, FIG. 5C and FIG. 6C, FIG. 5C is a partial schematic view of an automated nucleic acid extracting apparatus according to a preferred embodiment of the present invention, and FIG. 6C is a top view of a cassette of the automated nucleic acid extracting apparatus according to another preferred embodiment of the present invention. As shown in fig. 5C and 6C, the abutting portion 314 has a polygonal wall, which may include at least two supporting walls, and a structure including a first supporting wall 3142 and a second supporting wall 3143 is used for illustration, and the first supporting wall 3142 and the second supporting wall 3143 form an included angle e smaller than 180 degrees. Preferably, the included angle e can be 150 degrees; preferably, the included angle e can be 120 degrees; preferably, the included angle e can be 90 degrees; preferably, the included angle e may be 45 degrees as long as the pipe column discharge pipe 7 can abut against and stand. Specifically, please refer to FIGS. 6D to 6H, which illustrate various embodiments of the engaging portion of the cassette of the automated nucleic acid extracting apparatus of the present invention. The abutting portion 314 is, for example and without limitation, a circular arc-shaped wall 3141 (as shown in fig. 6E) or a polygonal wall (as shown in fig. 6D, 6F to 6H), the polygonal wall is, for example and without limitation, a structure having two supporting walls, three supporting walls, four supporting walls, five supporting walls, six supporting walls, or more than six supporting walls, and each supporting wall has an included angle therebetween, and the number of the supporting walls and the included angle are not limited as long as the column row suction pipes 7 can abut against and stand. In particular, as shown in fig. 6D, both ends of the polygonal wall of the abutting portion 314 may directly meet the parallel wall 31a, however, as shown in fig. 6F to 6H, both ends of the polygonal wall of the abutting portion 314 of the vertical wall 31b do not directly meet the parallel wall 31a, but meet the parallel wall 31a by the vertical wall 31b, without limitation.

Due to the design of the abutting portion 314, the abutting portion 72 of the column pipette 7 abuts against the arc-shaped wall 3141 of the abutting portion 314 or between the first support wall 3142 and the second support wall 3143 in the process of discharging liquid (for example, reactant, washing buffer solution, etc.), so that the column pipette 7 does not fall off and separate from the syringe 5 due to excessive pressure when the syringe 5 discharges liquid. Even if the column discharge pipette 7 is released when the syringe 5 is raised after discharging the liquid, the abutting portion 72 of the column discharge pipette 7 abuts and stands on the abutting portion 314 without completely separating from the syringe 5 by the design of the abutting portion 314, and then the syringe 5 and the column discharge pipette 7 can be tightly fitted again by the subsequent step of moving the syringe 5 up and down in the direction L2 perpendicular to the linear direction L1, so as to facilitate the subsequent extraction step.

Referring to FIG. 1, FIG. 6A and FIG. 6B, FIG. 6B is a top view of another preferred embodiment of the cassette of the automated nucleic acid extracting apparatus of the present invention. In the cartridge of FIG. 6B, the vertical wall 31B between the wash solution tank 312 and the eluate tank 313 has a completely hollow cylindrical structure, forming a filter accommodating space 315 for accommodating a filter. In detail, when the biological sample is tissue or other samples with impurities, the filter material is used to filter the dissolved products to fix the tissue fragments or impurities outside the filter material, and the clarified (without tissue fragments or impurities) dissolved products can be sucked into the sample suction pipe 6 to prevent the tip 62 of the sample suction pipe 6 from being blocked by the tissue fragments or impurities. By the design, the tissue sample can be directly loaded on the machine, and a centrifuge is not needed to be used for centrifugation outside the machine, so that a clarified lysate is obtained and loaded on the machine. Therefore, the step of cracking the tissue sample can be completed on the automatic nucleic acid extraction device of the invention, so that the tissue sample can also achieve the effect of full-automatic nucleic acid purification. Particularly, when extracting ribonucleic acid (RNA) from a sample, the filter material accommodating space 315 may further accommodate an enzyme required in the middle of the extraction process, such as deoxyribonucleic acid (DNase), to remove DNA (DNA) in the sample during the extraction process, and the DNase may also be washed and removed by the subsequent washing step, so that ribonucleic acid (RNA) without DNA (DNA) can be obtained after the extraction is completed.

Referring to fig. 1 and 2, in this example, the movable frame 4 and the syringe 5 perform a linear movement L1 (i.e., the syringe moves and the cassette does not move relative to the table), so the depth of the machine is only the length D3 of the seat B plus the thickness D4(D3+ D4) of the movable frame 4. However, in the prior art, the holder body B is linearly moved, and in order to enable the syringe to suck the solution in each accommodating space, the depth of the machine must be at least 2 times the length D3 of the holder body B plus the thickness D4(D3 × 2+ D4) of the movable frame 4. For example, the length D3 of the seat B of the automated nucleic acid extracting apparatus of this example is, but not limited to, 30 cm, the thickness D4 of the movable rack 4 is, but not limited to, 20 cm, and the depth of the machine is, for example, but not limited to, about 50 cm, thereby saving the user's space.

Referring to FIG. 9 in conjunction with FIGS. 1 to 3C, an embodiment of the present invention is described, wherein the automated nucleic acid extraction method is performed by using the aforementioned automated nucleic acid extraction apparatus, and a driving roller belt is disposed at the bottom of the automated nucleic acid extraction apparatus, and drives the moving rack 4 to move along the linear direction L1. In the preferred embodiment, the automated nucleic acid extraction method comprises the following steps: step S05 is performed by sucking the mixed reactant in the binding buffer containing space with the syringe 5 and the column pipette 7, so that the nucleic acid in the reactant is bound to a membrane 74 in the column pipette 7, and the reaction residue is discharged to the cassette 31, so that the nucleic acid in the reaction residue is bound to the membrane 74. Then, in step S06, the syringe 5 is used with the column pipette 7 to suck up the washing buffer solution in at least one washing solution tank through the membrane 74, and the syringe 5 is used with the column pipette 7 to discharge the washing buffer solution through the membrane 74. Next, step S08 is performed to aspirate the elution buffer from the eluate chamber through the membrane 74 with the syringe 5 engaging with the column discharge tube 7, and to discharge the elution buffer with nucleic acids to the collection tube 8 through the membrane 74 with the syringe 5 engaging with the column discharge tube 7. In addition, a step S07 may be arranged between the steps S06 and S08 to move the column pipette 7 to the column pipette holding space 22 for heating to minimize the residual amount of the washing buffer on the membrane 74. The steps S05 to S08 are performed by an automated nucleic acid extraction apparatus, and the dotted line shown in FIG. 9 (steps S01 to S04) may be performed manually by a user outside the automated nucleic acid extraction apparatus or may be performed entirely by the automated nucleic acid extraction apparatus. For example, a user can first dissolve and centrifuge a sample, then mix the supernatant with the binding buffer, add the mixture into the binding buffer containing space, and perform the subsequent steps with an automated nucleic acid extraction device; or, the user can dissolve and centrifuge the sample, add the supernatant into the containing space of the binding buffer solution to mix with the binding buffer solution, and then carry out the subsequent steps by the automatic nucleic acid extraction device; alternatively, the user can first dissolve and centrifuge the sample, add the lysate to the lysis solution tank, and perform the subsequent steps by the automated nucleic acid extraction apparatus, which is not limited in the present invention.

In this embodiment, to illustrate the case where the steps S01 to S08 are performed by the automated nucleic acid extracting apparatus, in the steps S01 to S08, the automated nucleic acid extracting apparatus and the moving rack 4 (driving the syringe 5) move to a specific position along the linear direction L1 to perform the above steps, which are described in detail as follows: before step S01, the syringe 5 disposed on the movable rack 4 is moved to the sample discharge pipette receiving space 21 of the column receiving area 2 in the linear direction L1, so that the syringe 5 is connected to the sample discharge pipette 6 received in the sample discharge pipette receiving space 21 and the sample discharge pipette 6 is taken out from the sample discharge pipette receiving space 21. Next, the sample discharge pipette 6 connected to the syringe 5 is moved to the cassette 31 in the cassette receiving area 3 in the linear direction L1. Then, in step S01, the lysis buffer is sucked from the lysis solution tank 311 of the cartridge 31, and then moved to the sample receiving area 1. Next, step S02 is performed to discharge the lysis buffer to the sample-holding space 11 of the sample-holding section 1 and mix the lysis buffer. Then, step S03 is performed to suck the mixed lysate in the sample-accommodating space 11 and move the lysate to the binding buffer-accommodating space 12 of the sample-accommodating section 1. Then, step S04 is performed to discharge the lysate to the binding buffer accommodating space 12 and mix with the binding buffer to perform the binding reaction. Then, before step S05, the syringe 5 and the sample discharge tube 6 are moved to the sample discharge tube accommodating space 21, the stripper plate 41 is driven by the spring mechanism 42 to move so that the sample discharge tube 6 is detached from the syringe 5 to the sample discharge tube accommodating space 21, and then the syringe 5 is moved to the column discharge tube accommodating space 22 of the column accommodating area 2 along the linear direction L1, so that the syringe 5 is connected to the column discharge tube 7 disposed in the column discharge tube accommodating space 22, and the column discharge tube 7 having the membrane 74 disposed therein is taken out from the column accommodating area 2. Thereafter, the column discharge pipette 7 connected to the syringe 5 is moved in the linear direction L1 to the binding buffer accommodating space 12. Next, in step S05, the syringe 5 is used with the column discharge pipette 7 to aspirate the formed reaction product, so that the reaction product passes through the membrane 74 and the nucleic acid contained in the reaction product is attached to the membrane 74. Next, the syringe 5 is moved to the cassette 31 in the linear direction L1. Then, the reaction product is passed through the membrane 74 again in the direction of gravity to attach the nucleic acid contained therein to the membrane 74, and the reaction residue is discharged to the cassette 31 in the direction of gravity, and the reaction residue is passed through the membrane twice to ensure that the nucleic acid in the reaction product is attached to the membrane 74. Next, in step S06, the column pipette 7 with nucleic acid is moved along the linear direction L1 along the syringe 5 and the moving frame 4 to the washing solution tank 312 filled with washing buffer solution and then washed by sucking and discharging the washing buffer solution, so that the washing buffer solution passes through the membrane 74 twice to wash away the reaction residue remaining on the membrane 74. After washing, the column pipette 7 with the nucleic acid can be moved to the column pipette receiving space 22 along the linear direction L1 along with the syringe 5 and the moving frame 4, the column pipette 7 is retained in the column pipette receiving space 22, and heated by the heating element H to evaporate the liquid (e.g., washing buffer) remaining on the membrane 74 of the column pipette 7, so as to dry it sufficiently (i.e., step S07). Finally, the nucleic acid-carrying column discharge pipette 7 is moved along the linear direction L1 with the cylinder 5 and the moving frame 4 to the eluent tank 313 filled with the elution buffer and elution is carried out by sucking and discharging the elution buffer at step S08, thereby obtaining an eluate (eluate, elution buffer with nucleic acid). The column pipette 7 then aspirates the resulting eluate, which then travels in a linear direction L1 to the collection tube 8, and discharges the eluate to the collection tube 8. In particular, the heating member H is heated for a time such as, but not limited to, 30 seconds, 1 minute, 3 minutes, 5 minutes as long as the liquid remaining on the membrane 74 of the column evacuation tube 7 is volatilized and sufficiently dried, and the present invention is not limited thereto.

In the preferred embodiment, in the step of discharging the reaction residue to the cassette 31 (step S05) and the step of discharging the washing buffer from the syringe 5 with the column discharge pipette 7 through the membrane 74 (step S06), the abutting portion 72 of the column discharge pipette 7 abuts against the abutting portion 314 of the cassette 31, and then the reaction residue or the washing buffer is discharged. By the design of the abutting part 314, when the syringe 5 discharges liquid, the column discharge straw 7 abuts against the abutting part 314, so that the column discharge straw 7 cannot fall off from the syringe 5 and separate due to excessive pressure in the process of discharging liquid. Even if the column discharge pipette 7 is released when the syringe 5 rises after discharging the liquid, the abutting portion 72 of the column discharge pipette 7 abuts and stands on the abutting portion 314 without being completely separated from the syringe 5 by the design of the abutting portion 314, and the syringe 5 and the column discharge pipette 7 can be tightly fitted again by the subsequent steps.

In the preferred embodiment, after the step of abutting the abutting portion 72 of the pipette 7 against the abutting portion 314 of the cassette 31 and then discharging the reaction residue or the washing buffer (steps S05, S06), the method further comprises the following steps: the abutting part 72 of the column discharge pipette 7 abuts on the abutting part 314 of the magazine 31, and the syringe 5 is moved up and down by 5 mm or less in the direction L2 perpendicular to the linear direction L1. Through this step, the column discharge tube 7 can be tightly connected with the syringe 5, preventing the column discharge tube 7 from loosening and falling. Preferably, the needle cylinder 5 can move up and down 5 mm in the direction L2; preferably, the needle cylinder 5 can move up and down 4.5 mm in the direction L2; preferably, the syringe 5 is moved up and down 3 mm in the direction L2 to bring the column discharge pipette 7 into close contact with the syringe 5 again.

In the preferred embodiment, the number of the washing liquid tanks 312 can be adjusted according to the actual requirement of the user, and the invention is not limited thereto. In detail, if the number of the washing liquid tanks 312 is more than one, before moving the column pipette 7 to the column pipette holding space 22 for heating, the steps of sucking at least one washing buffer in the washing liquid tank by the syringe 5 with the column pipette 7 through the membrane 74, and discharging the washing buffer by the syringe 5 with the column pipette 7 through the membrane 74 may be repeated at least once. That is, the steps of moving the column pipette 7 with the nucleic acid along the linear direction L1 to the washing solution tank 312 containing the washing buffer along the syringe 5 and the moving rack 4 and performing washing by repeatedly sucking and discharging the washing buffer can be performed in different washing solution tanks 312, and the number of washing steps is determined according to the number of the washing solution tanks 312.

In the preferred embodiment, the method further includes the step of moving to the filter material accommodating space 315 and combining the sample discharge tube 6 associated with the syringe 5 and the filter material in the filter material accommodating space 315, between the step of discharging the lysis buffer to the sample accommodating space 11 and mixing the lysis buffer (step S02) and the step of sucking the mixed lysate in the sample accommodating space 11 (step S03); and a step (step S03) of moving the sample suction pipe 6 and the filter medium to the solution tank 311 and reciprocating the sample suction pipe and the filter medium in the solution tank 311 in a linear direction after the step of sucking the dissolved product mixed in the sample-accommodating space 11. Specifically, when the biological sample is tissue or other sample with impurities, the filter medium is used to filter the dissolved product after dissolution, so as to clamp the tissue fragments or impurities on the outer side of the filter medium, and then step S03 is performed to suck the clarified (without tissue fragments or impurities) dissolved product into the syringe 5. Then, the sample suction pipe 6 and the filter material are moved to the dissolution liquid tank 311 and reciprocated in the dissolution liquid tank 311 along the linear direction, so that impurities clamped and fixed on the outer side of the filter material are cleaned, and then the dissolution product in the needle cylinder 5 is discharged to the combination buffer solution accommodating space 12 for combination reaction, thereby avoiding the influence of tissue fragments or impurities on the effect of the combination reaction and further on the extraction efficiency. That is, the foregoing steps may be added when the biological sample is tissue or other sample with impurities.

In the preferred embodiment, the number of the sample accommodating space 11, the combination buffer accommodating space 12, the sample discharging and pipette accommodating space 21, the column discharging and pipette accommodating space 22, the solution tank 311, the washing solution tank 312, the eluent tank 313, the abutting portion 314, the syringe 5, the sample discharging and pipette 6, the column discharging and pipette 7, the collection tube 8 and the connector 51 can be adjusted by the user according to the actual requirement, and the invention is not limited thereto. Specifically, the cartridge 31, the sample accommodating space 11, the binding buffer accommodating space 12, the sample discharge/pipette accommodating space 21, the column discharge/pipette accommodating space 22, the syringe 5, and the collection tube 8 are arranged along the linear direction L1, and the order of arrangement is not limited herein.

In summary, the automated nucleic acid extracting apparatus of the present invention has the design that each of the solution tank 311, the wash tank 312 and the adjacent eluent tank 313 is provided with the abutting portion 314, so that the user can avoid the reactant from splashing out of the solution tank 311, the wash tank 312 or the eluent tank 313 during the automated nucleic acid extraction, and can also avoid the column discharge pipette 7 from loosening and falling off. In addition, by the design that the cassettes 31 and the accommodating spaces are arranged along the linear direction L1 and the moving rack 4 and the syringe 5 repeatedly move along the linear direction L1, the effect of automatically extracting nucleic acid in the linear direction L1 can be achieved, thereby avoiding sample contamination and improving the extraction efficiency. Therefore, the automated nucleic acid extraction apparatus of the present invention can extract nucleic acids, such as cfDNA and ctDNA, with high yield and high concentration from the sample quickly and conveniently.

The foregoing is by way of example only, and not limiting. It is intended that all equivalent modifications or variations not departing from the spirit and scope of the present invention be included in the claims.

Claims

1. An automated nucleic acid extraction apparatus, comprising:

the base body is provided with a sample accommodating area, a pipe column accommodating area, a cassette accommodating area and a collecting pipe, and the sample accommodating area, the pipe column accommodating area, the cassette accommodating area and the collecting pipe are arranged along a linear direction;

a cassette, disposed in the cassette receiving area, the cassette including two parallel walls and at least two vertical walls, the parallel walls and the vertical walls forming a solution tank, at least one wash solution tank and an eluent tank together, wherein the vertical walls forming the solution tank, the wash solution tank and the eluent tank are respectively disposed with an abutting portion, and the solution tank, the wash solution tank and the eluent tank are also arranged along the linear direction, wherein the abutting portion has a circular arc wall or a polygonal wall;

the driving unit is arranged on the seat body;

the moving frame is vertically arranged on the seat body and driven by the driving unit to reciprocate along the linear direction;

and the needle cylinder is arranged on the moving frame and moves along with the moving frame.

2. The automated nucleic acid extraction apparatus of claim 1, wherein the sample receiving compartment comprises a sample receiving space and a binding buffer receiving space.

3. The automated nucleic acid extraction apparatus according to claim 1, wherein the column receiving section further comprises a sample row pipette receiving space and a column row pipette receiving space, and the automated nucleic acid extraction apparatus further comprises a sample row pipette and a column row pipette, and the sample row pipette and the column row pipette are movably disposed in the sample row pipette receiving space and the column row pipette receiving space.

4. The automated nucleic acid extraction apparatus of claim 3, wherein the syringe is detachably connected to the sample discharge pipette or the column discharge pipette.

5. The automated nucleic acid extraction device of claim 1, wherein an arc angle of the circular arc-shaped wall is greater than or equal to 90 degrees when the abutment has the circular arc-shaped wall; when the abutting part is provided with the polygonal wall, the polygonal wall comprises at least two supporting walls, and the supporting walls mutually form an included angle of less than 180 degrees.

6. The automated nucleic acid extracting apparatus according to claim 1, wherein the vertical wall between the wash solution tank and the eluate tank has a complete hollow cylindrical structure, forming a filter accommodating space.

7. The automated nucleic acid extraction apparatus of claim 1, wherein a recess is formed in a bottom of the eluate chamber, and the cartridge further comprises a snap member.

8. An automated nucleic acid extraction method applied to the automated nucleic acid extraction apparatus according to any one of claims 1 to 7, wherein the automated nucleic acid extraction method comprises the following steps:

sucking a mixed reactant in a containing space of a combining buffer solution by matching the needle cylinder with a column suction pipe, combining nucleic acid in the reactant with a diaphragm in the column suction pipe, discharging reaction residues to the cassette, and combining the nucleic acid in the reaction residues with the diaphragm;

sucking the washing buffer solution in the at least one washing solution groove by the needle cylinder and the column suction pipe through the diaphragm, and then discharging the washing buffer solution by the needle cylinder and the column suction pipe through the diaphragm;

and sucking the elution buffer solution in the elution liquid groove by matching the needle cylinder with the column suction pipe through the membrane, and then discharging the elution buffer solution with nucleic acid to the collection pipe by matching the needle cylinder with the column suction pipe through the membrane.

9. The automated nucleic acid extraction method of claim 8, wherein in the step of discharging the reaction residue into the cassette and the step of discharging the washing buffer with the syringe and the column pipette through the membrane, the abutting portion of the column pipette is abutted against the abutting portion of the cassette, and then the reaction residue or the washing buffer is discharged.

10. The automated nucleic acid extraction method of claim 9, further comprising, after the step of abutting the abutting portion of the column pipette against the abutting portion of the cassette and then discharging reaction residues or wash buffer, the steps of:

and abutting the abutting part of the pipe column row suction pipe against the abutting part of the cassette, and moving the needle cylinder up and down in a direction vertical to the linear direction by less than or equal to 5 mm.